Multiwell stat test

ABSTRACT

An apparatus is provided for the detection and semi-quantitative measurement of analytes. The assay results are visualized by the formation on a filter of a colored annular or circular spot, the diameter of the spot being related to the concentration of the analyte of interest. The filter on which the assay results are visualized is divided into multiple regions by strips of non-porous tape crossing the filter surface. The invention also includes a component for diluting sample to a suitable concentration for analysis, and dispensing the diluted sample onto the test filter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 644,941, filedJan. 23, 1991, now U.S. Pat. No. 5,147,780, which application is adivision of application Ser. No. 444,814, filed Dec. 1, 1989, nowabandoned.

INTRODUCTION

1. Technical Field

The field of this invention relates to assay devices employing animmobilized specific binding pair member and for collecting, dilutingand dispensing clinical specimens.

2. Background

Many immunoassay procedures have been devised for the purpose ofdetecting specific analytes. Such assays have found countlessapplications as tools in medicine. Analyte specific assays have beenused to detect antibodies produced in response to infection, componentsof pathogenic agents, levels of drugs, hormones, and enzymes, etc. Inaddition to medicine, immunoassays and other related assays have alsofound numerous applications in manufacturing industries, for example,the detection of food contaminants.

Heterogeneous immunoassays usually involve a ligand or antibodyimmobilized to a solid support. A sample containing the analyte ofinterest is passed over the immobilized immunoreagent and the amount ofantibody-ligand complex formed is measured. In heterogenous assays,essential elements include the anchoring of one member of a specificbinding pair to a solid support, and a means for either directly orindirectly detecting label bound to the support.

The ease of performing an assay procedure is always an importantconsideration. Most assays involve the addition of multiple reagents andrequire multiple washing steps. Ideally, an assay will be simple and notrequire the use of complex equipment such as microtiter plate washers orELISA readers. Immunoassays able to be performed in a physician'soffice, at home, or in the field are of particular interest and must bedeveloped to be performed without the use of specialized equipment.

Numerous immunoassays exist in which the results are visualized by theformation of a colored spot. The intensity of the color of a spot isusually proportional to the concentration of analyte in the sample andrequires instrumentation to relate the color intensity to analyteconcentration. Otherwise, with visual comparison of the color intensityof spots produced by different samples may be an uncomfortablysubjective exercise. Even with semiquantitative assays, differences maybe only difficultly distinguished. Furthermore, analysis of test resultsthat exclusively rely on color changes may be exceptionally difficultwhen weakly positive results are obtained. There is therefore interestin providing semi-quantitative assays which have simple protocols, andsubstantially reduce subjective error associated with the determinationof a positive result and the amount of analyte.

3. Relevant Literature

U.S. Pat. Nos. 4,727,019 and 4,632,901 relate to immobilized phaseimmunoassay devices that produce a colored spot when exposed to a samplecontaining appropriate analyte for detection.

SUMMARY OF THE INVENTION

Methods and apparatus are provided for performing a non-instrumentalassay for the detection of an analyte in a liquid sample. The apparatuscomprises a porous reactive filter, a porous separation layer, anon-porous flow control layer and an absorbent waste fluid receivinglayer. The reactive filter comprises a specific binding pair memberdistributed in a radial non-linear concentration gradient. A circular orannular shaped spot is produced on the porous reactive filter, where asmall diameter spot indicates analyte concentration below apredetermined level.

A sample preparation apparatus is also provided that serves as acollector-diluter-dispenser comprising a cap enclosed tube. Thecollector-diluter-dispenser device absorbs sample by means of a nib. Thecollector-diluter-dispenser contains a liquid medium restrained from thenib by a frangible barrier which supports the nib, with the nibextending through an opening in the cap. After absorbing the sample withthe nib, breaking the barrier drops the nib into the liquid medium andthe sample dissolves into the liquid medium. Exerting pressure on thesides of the collector-diluter-dispenser permits the diluted sample tobe dispensed through the opening originally containing the nib and ontothe assay apparatus.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the followingdetailed description of specific embodiments when considered incombination with the drawings that form part of this specification,wherein:

FIG. 1 is an obliquely positioned perspective view of an assayapparatus;

FIG. 2 is a plan view of the porous reactive filter of the apparatus ofFIG. 1;

FIG. 3 is an elevational cross-sectional view of thecollector-diluter-dispenser apparatus; and

FIG. 4 is an elevational cross-sectional view of an alternate embodimentof the collector-diluter-dispenser apparatus of FIG. 3.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The system of the subject invention comprises two principal components.The first component as shown in FIG. 1 is an apparatus for assaying thepresence of an analyte of interest. The second component as shown inFIGS. 3 and 4, consists of a device for diluting sample and dispensingthe diluted sample. The second component can be used to apply thediluted sample to the assay apparatus.

The assay apparatus of FIG. 1 is used to detect the presence of ananalyte in a sample. The assay apparatus is able to semi-quantitativelymeasure the amount of the analyte found in the sample. Analytes suitablefor detection in the assay apparatus are members of specific bindingpair members. Specific binding pairs are defined as two non-identicalmolecules capable of specifically and usually non-covalently binding toeach other in solution so as to form stable complexes that can bedetected either directly or indirectly. Exemplary but not exclusive ofgeneral classes of specific binding pair interactions areligand-receptor interactions, which are primarily exemplified byantibody-hapten or antibody-antigen interactions. Ligands for the mostpart will be non-proteinaceous, naturally occurring or synthetic organicmolecules of from about 125 to 5,000 Dal, and peptides and proteins.Receptors that may be detected by the subject apparatus will for themost part be proteins, such as immunoglobulins, fragments thereof,particularly monovalent fragments, of immunoglobulins, e.g., Fab, Fv,etc., enzymes, naturally-occuring receptors, e.g., T-cell receptors,hormone receptors, surface membrane receptors, lectins, etc. Otherspecific binding pairs include nucleic acids, e.g., DNA and RNA. For adisclosure of specific ligands and receptors see U.S. Pat. No.3,996,345, columns 10-17, which disclosure is herby incorporated byreference.

Results from assays performed with the subject apparatus are visualizedas an annular or circular spot formed on a filter. The spot for apositive result has a dark central region and a lighter exterior region.Differences in concentration can be detected by having a dark centralring or both the central dark ring and a colored outer ring. As detectedin the subject invention, the correlation between the diameter of theresult and indicator spot and the analyte concentration providessignificant advantages. By providing for a small high intensity spot foranalyte within a predetermined concentration, and a larger, less intensespot above such concentration visual detection can give a reasonableestimate of the amount of analyte, particularly whether below or above athreshold value.

The correlation between the diameter of the indicator spot and theconcentration of analyte is achieved by immobilizing a binding pairmember to the porous filter in a non-linear radial concentrationgradient, which may be the same compounds as the analyte, across-reactive compound, or a reciprocal binding pair member. The term"reciprocal binding pair member" is intended to mean the member of aspecific binding pair which complexes with the designated member,frequently in reference to the analyte as the designated member. Theradial concentration gradient is arranged so that the highestconcentration of the specific binding pair member is at the inner regionof a ring, having a relatively small diameter relative to a secondconcentric ring comprising a lower concentration per unit area of thesame binding pair member. Usually the central ring will have a diameterin the range of 0.1 to 0.5:1, usually 0.1 to 0.3:1 as compared to thediameter of the outer ring.

As shown in FIG. 1, the assay apparatus contains multiple layers,arranged in a specific order and held together in register by a housing7. The assay apparatus has four principal layers. The layers willusually be of essentially the same circumferential dimensions, e.g.,length and width, but may vary with respect to one another as to thethickness. The principal layers, in descending order, are as follows.The top layer is a porous reactive filter 3, which may be divided intoregions by a non-porous divider, e.g., tape 9; at least one of theregions, usually all or most of the regions, contain at least onespecific binding pair member ring 10 capable of forming complexesrelated to the analyte. Beneath and contacting the filter layer is aporous separation layer 4. Below the porous support layer is a flowratecontrol layer 5. The bottom layer is a waste fluid receiving absorbentpad 6. The porous separation layer 4, the flow rate control layer 5, andthe waste fluid receiving pad 6, may be excluded, but will normally bepresent.

By reactive in referring to the porous reactive filter, it is intendedthat immobilized to this filter is a specific binding pair membercapable of binding the analyte or its reciprocal binding member. Theporous reactive filter may be composed of paper, cellulose, glass fiber,nylon, PVDF, or the like. Preferably the filter will be comprised ofnylon. Commercially available examples of such filters include Immobilon(Millipore), Meantest membrane (memtek), Biodyne (Pall), Immunodyne(Pall), and Ultrabind (Gelman Sciences). The pores in the porousreactive filter will have an average diameter in the range of about 0.1μto 10μ, usually in the range of about 1μ to 7μ.

The porous reactive filter 3 may be divided into a plurality of regions,conveniently separated by non-porous dividers, e.g., tape 9. Thedifferent regions may serve different functions, for example, providingfor different concentration ranges of the specific binding pair memberor different binding pair members for panel test applications. A controlmay have a predetermined amount of label or be free of any binding pairmember or the like. All or a portion of the region may contain thespecific binding pair member. The regions will conveniently be in therange of from about 3 to 50 mm², usually 4 to 14 mm². The non-poroustape can provide a color contrast with the regions, preferably beingwhite or yellow to enhance the colored appearance of a positive result.The separation between regions provided by the tape will generally beabout 0.2 to 15, usually 1 to 4 mm.

A member of the specific binding pair either binding to orcross-reactive with analyte is applied and becomes non-diffusively boundor immobilized to a porous filter so as to generate a circular spot orring with a non-linear radial concentration gradient that has asubstantial drop in concentration at a relatively short distance fromthe center of the ring, generally dropping by at least about twenty fivepercent within a band of about 0.5 mm width from the averageconcentration of the central region. The filter material employed isdesirably, but not necessarily, chemically reactive so as to covalentlybond the specific binding member. By appropriate application of thebinding pair member solution, a high concentration of binding pairmember can be obtained in a small radius from the center surrounded by aconcentric contiguous outer circle of larger radius and substantiallylower concentration.

The application solution will normally be a buffered solution at a pH inthe range of about 4 to 10, with a concentration of specific bindingpair member of about 10 μg/ml to 5 mg/ml. Other solutes may include saltat a concentration in the range of about 10 μM to 1M. By lowering orincreasing the buffer concentration or adding other unreactive solutes,e.g., glycols, the rate of diffusion of the specific binding pair membermay be modified to increase or decrease the diameter of the highconcentration region.

The high concentration region is achieved by virtue of the highreactivity of the porous filter, compression in the region about thesite of application of the solution and depletion of the specificbinding pair member from the solution in the central region.

The nib can be applied to the porous reactive filter at a pressure whichmodifies the porosity of the membrane in the depressed area, so as to besufficient to produce a ring of binding pair member about an uncoloredcenter, rather than a completely filled circle.

Different binding pair members may be applied to different regions sothat the presence of multiple analytes in a single sample may besimultaneously analyzed. The same binding pair members at differentconcentrations may be applied to different regions so as to aid in thequantitative determination of the analyte concentration and provide fora wider range of detectable analyte concentrations on a single orsubdivided porous reactive filter or the individual regions may beindependent filter elements. One or more spots of the same binding pairmember may be applied per region, normally at the same concentration.When more than one binding pair member spot is present in a singleregion, the spots may be overlapping. In a preferred embodiment of theinvention, two binding pair member spots will be present in eachmeasurement region. One or more regions may not contain a binding pairmember so as to provide a negative control. Positive controls may alsobe provided for analyte by providing for the presence of a predeterminedamount of label.

A porous separation layer 4 is located immediately beneath, directlycontacting, and in register, with the porous reactive filter layer 3 ofthe assay apparatus. The porous separation layer 4 is in contact withthe lower surface of the porous reactive filter 3. The porous separationlayer serves to support the porous reactive filter and permit reagentsto flow uniformly from the top layer down to lower layers of the assayapparatus. The porous separation layer may be made of any rigid orsemi-rigid porous material that does not substantially bind or interactwith reagents used in conjunction with the invention. Exemplary ofmaterials for the porous separation layer are fiberglass, paper,hydrophilic polypropylene, and cellulose, preferably the porousseparation layer is made of H-HDC (Pall). The porous separation layer isof essentially the same circumferential dimensions or shape as theporous reactive filter layer or the elements thereof. The thickness ofthe porous separation layer will generally be in the range of about 0.1mm to 1 mm.

Immediately beneath and contacting the porous separation layer 4, isflowrate control layer 5. Conveniently, the flowrate control supportlayer 5 contains a plurality of small uniformly placed, linearly, orrandomly, arranged perforations, generally in one or two lines,preferably having the perforations below each region. The flowratecontrol layer serves to both slow and direct the flow of reagentsthrough the porous reactive filter 3. The flowrate control layer 5 maybe made from any non-porous wettable material that is substantiallyinert to the reagents employed in the performance of an assay. Theflowrate control layer will be of essentially the same circumferentialdimensions or shape as the filter layer. The precise thickness of theflowrate control layer is not essential to the function of the subjectinvention, generally ranging from about 2 to 10 mils.

The perforations will be of a size and number which serve to impede theflow of liquid reagents through the apparatus. In general, the greaterthe desired time of contact between the porous reactive filter and thereagents, the smaller the cross-sectional area of the perforations willbe. The shape of individual perforations is not important. Individualperforations will usually have a cross-sectional area in the range ofabout 5 mils to 50 mils, more usually 7 mils to 15 mils with the numberof perforations being about 1 to 10 per cm along the length of theflowrate control layer. The holes may be uniformly distributed so as topermit a uniform flow of reagents through the different porous regionsof the porous reactive filter layer 3. It is preferable, though notessential, that the perforations in the flowrate control layer belocated beneath the tape free regions of the porous reactive filter 3.

Below and directly contacting the flowrate control layer 5, is a wastefluid receiving layer 6. Reagent solution flowing through perforationsin the flow rate control layer directly enter the waste fluid receivinglayer. The waste fluid receiving layer draws reagent solutions away fromthe other layers of the assay apparatus. The absorbing volume of thewaste fluid receiving layer is substantially greater than the totalvolume of reagents required to be added to the assay apparatus for theperformance of a given assay. The waste fluid receiving layer may be ofany convenient material, such as cotton, blotting paper, polyesterfibers, cellulose acetate, or the like.

The multiple layers of the assay apparatus are held together in anapparatus housing 7. The housing will be made of an inert materialconveniently being any of a variety of commercial plastics which may bemolded, for example, polyethylene, polypropylene, styrene, ABS,polyacrylate, polystyrene, or the like.

FIG. 1 illustrates one possible embodiment of such a housing. Thehousing is capable of compressing the layers to maintain continuous anduniform contact between the layers of the apparatus so that liquidflowing through the apparatus will flow uniformly through the porousreactive filter 3. The housing may consist of one or more pieces.Preferably, the housing will consist of two pieces, an upper reservoir1, and a lower casing 7. The reservoir 1 is the top portion of thehousing and may have an inner lip to maintain the layers undercompression. For convenience, the reservoir may be separable from therest of the housing.

The reservoir may be partially or completely filled with the dilutedsample, so that diluted sample is uniformly distributed over the filtersurface. The reservoir may contain marking lines that indicate theamount of solution added. The reservoir has an open bottom 2 and isenclosed at the bottom with porous reactive filter 3. The apparatus maybe marked so as to distinguish one end of the apparatus from the other.The markings may be inherent in the shape of the housing by making thehousing asymmetric along at least one axis, or the polarity markings maybe manifested as symbols present on the housing.

The precise dimensions of the housing are not essential to the functionof the assay apparatus, but in general, the apparatus will be of a sizeconvenient for transport, manipulation, and assembly. The housing willgenerally have a length in the range of about 0.5 to 5 cm, preferably inthe range of about 2 to 5 cm. The width will be in the range of about0.3 to 3 cm, preferably in the range of about 0.5 to 1 cm. Preferably,the width will be about 9 mm so as to allow batch testing using astandard laboratory multichannel micropipetter. The height of thehousing will be in the range of about 0.5 to 5 cm, preferably in therange of about 1 to 3.5 cm.

A collector-diluter-dispenser apparatus depicted in FIGS. 3 and 4 may beused either in conjunction with, or independently of, the firstcomponent of the subject invention, the assay apparatus (FIG. 1). Thecollector-diluter-dispenser apparatus comprises five principalcomponents: a hydrophilic nib 11, a cap 14 for retaining the nib fromfalling out of the collector-diluter-dispenser apparatus, a flexibletubular container 15, enclosed by cap 14, liquid medium 17, and afrangible barrier 16 restraining the flow of the liquid medium andsupporting the nib from falling into the liquid medium, while the nib 11extends through an aperture 18 in the cap 14.

The use of the collector-diluter-dispenser is described as follows.Liquid sample is contacted with the nib 11 of thecollector-diluter-dispenser. Once the sample has been absorbed by thenib, the nib is withdrawn from the sample source, and the nib end of thecollector-diluter-dispenser is pointed upwards. The frangible barrier 16within the device is broken, and the breaking of the barrier 16 allowsthe sample containing nib 11 to fall into the liquid medium 17 where thesample is released from the nib 11 and dispensed into the liquid medium17. The nib is capable of absorbing molecules, particles, e.g., virusparticles, cells, etc., and effectively dispersing them into the liquidmedium. After the liquid medium and sample have mixed, thus diluting thesample, the collector-diluter-dispenser is used as a dropper to dispensethe diluted sample into the previously described assay apparatus. Thecollector-diluter-dispenser may be used as a dropper because the maintube 15 is made from a flexible material that deforms under pressure,and diluted sample is free to flow through an aperture 18 in the capthrough which the nib 11 had previously extended.

The nib 11 serves several functions. The nib is able to absorb sample,and also release the absorbed sample into the liquid medium 17. The nibcan also absorb proteins and cells for release into the liquid medium17. Furthermore, the nib has a measuring function. Essentially identicalnibs will be able to take up and release reproducible quantities ofsample so that predetermined dilution ratios may be reproduciblyattained.

The nib 11, may also serve an active role by providing for variousreagents. The nib 11 may also include in dehydrated form, specimen oranalyte reactive compounds such as anti-coagulants (EDTA, citrate,heparin), detergents, etc. The nib 11 may also contain attached ligandsin dehydrated form so that the nib 11 may serve as a solid phase supportfor ligand analyte interactions.

Once the nib 11 has been used to collect sample, the sample may beallowed to dry on the nib 11 prior to the release of the sample into theliquid medium, or the sample containing nib may be mixed with the liquidmedium before substantial drying can take place.

The nib 11 will usually be composed of a hydrophilic relativelydeformable resistant material that will be substantially inert to theanalyte of interest. The nib is made of a hydrophilic material so thatan aqueous sample will be drawn up the nib when the nib is touched to afluid sample. Exemplary, but not exclusive of material suitable for thenib are nylon, polyethylene, or polypropylene. Preferably the nibmaterial is nylon. Preferably the nibs are Nib 99356 produced byAmerican Filtrona. The nib is essentially cylindrically shaped with theexposed end of the nib being pointed. The pointed end 12 of the nib iscontacted with the sample to be analyzed. The opposing end of the nibhas a bulbous shape 13 having a diameter somewhat larger than thediameter of the aperture 18 of the cap 14.

The nib will usually have a length of about 3 mm to 4 cm and a diameterof about 0.2 to 3 mm, depending on the sample size to be employed.

The cap 14 is mounted onto a flexible tube 15 sealed on one end 19. Theflexible tube is made of a material that readily deforms undersqueezing. Exemplary of materials for the tube are plastics such aspolyethylene, polypropylene, or other inert elastomeric materials. Theprecise dimensions of the tube are not critical, the tube convenientlyhaving a volume of about 1 to 5 ml.

The barrier 16 is made of a frangible material, preferably glass orplastic, and is of a thickness such that it is easily broken under handpressure. The material should be inert to both the liquid medium and thesample. There are many possible configurations of the barrier 16 thatpermit it to both support the nib and restrain the flow of liquidmedium. Two possible configurations are given in FIGS. 3 and 4. In FIG.3 the barrier 16 is the top of an ampoule containing the liquid medium.In FIG. 4 the barrier 16 is a disk extending completely across theflexible tube.

The composition of the liquid medium 17 will vary in accordance with therequirements of specific assays. In general, the liquid medium will bean aqueous solution. The liquid medium 17 may also contain compoundsthat have functions in the assay other than serving to dilute thesample. For example the liquid medium may contain buffers and/ornon-specific binding blocking agents, e.g., bovine serum albumin,casein, serum, etc. Liquid medium 17 may also contain a specific bindingpair member that may bind to an analyte contained in the specimen or achromogenic reagent.

The subject invention may be used with established immunoassayprocedures requiring the use of an immobilized phase. The use of theterm "immunoassay" is meant to comprise both immunoassays and assays ofsimilar design using immobilized specific binding pair members, eventhough neither member of the binding pair is an antibody or fragmentthereof. These procedures involve the addition of a variety of reagentsto detect the formation of specific binding pair complexes. Theformation of binding pair complexes will usually be detected by thepresence of a dye or fluorophore, which may be conveniently produced asa product of an enzyme mediated reaction. The labeled reagent is able tobind to binding pair complexes immobilized on the filter. Addition ofsuitable chromogenic reagents allows the enzyme label to reveal thelocation of the immobilized binding pair complexes by coloring theporous reactive filter.

The amount of sample and assay reagents added to the assay apparatus viathe reservoir varies with different embodiments of the subjectinvention. The reservoir may be filled to the top with diluted sampleand reagents, or lesser quantities may be added. In general, for a givenspecific embodiment, a predetermined and reproducible quantity ofdiluted sample will be added, while reagents will normally be present inexcess. The volume of diluted sample may be measured in drops from thecollector-diluter-dispenser apparatus or by a marker in the reservoir.One or more drops of diluted sample may be applied to each tape-freeregion, or the reservoir may either be filled partially or completely.Preferably the reservoir will be filled with diluted sample and reagentsso that uniform contact between the solution and the measurement regionsis maintained. Increasing the amount of diluted sample and reagent addedto the reservoir will increase the contact time between the addedsolution and the porous reactive filter as well as the amount ofspecific binding pair member which binds to the surface.

The following is an example of using the subject invention to assayblood for the presence of antibodies to HTLV-1. The amount of reagentsused will vary in accordance with the size of the apparatus. A drop ofblood obtained by a finger prick of a patient is placed on a glassslide. The tip of the nib 11 on the collector-diluter-dispenser istouched to the blood drop on the slide (or may be directly touched tothe pricked finger) and blood is drawn up the nib. When the nib issaturated, the collector-diluter-dispenser is then placed upright sothat the cap 14 is on top. The frangible barrier 16 is broken byexerting pressure on the walls of the collector-diluter-dispenser. Thenib falls into the liquid medium 17 and the collector-diluter-dispenseris then agitated to ensure proper mixing.

Several drops of diluted sample are then added to the reservoir on topof the assay apparatus so as to cover the reactive filter 3. The porousreactive filter contains spots of HTLV-1 envelope antigen in anon-linear radial concentration gradient bound to one or more of themeasurement regions; positive and negative controls are also presentwhere a known amount of label is present and no label is present,respectively. Immediately prior to the addition of diluted sample to thereservoir, a solution of blocking agent is added to the reservoir andthe reservoir is allowed to drain. By blocking agent it is intended asolution containing a compound or compounds, e.g., bovine serum albuminor casein, that will block any non-specific binding sites available onthe porous reactive filter. After addition of the diluted sample, thediluted sample is allowed to drain. A solution of biotinylated goatanti-human IgG antibodies are then added to the reservoir and thesolution allowed to drain. A solution containing streptavidin conjugatedalkaline phosphatase is then added to the reservoir and allowed todrain. A wash solution buffer is then added to the reservoir and allowedto drain. A solution containing a chromogenic alkaline phosphatasesubstrate is then added to the reservoir and allowed to drain. Aftercolor has developed, a reaction stop solution (0.2M phosphate pH 6) isadded to stop the enzyme reaction.

It is evident from the above description that the subject inventionprovides apparatuses and methods for performing a wide variety of assayssuitable for the detection and measurement of analytes in many types ofsample. The presence of an analyte is manifested by the formation of acolored spot. Once the concentration of analyte in a sample is above apredetermined threshold, the diameter of the colored spot increases todefine a larger diameter circle. The correlation between analyteconcentration and spot diameter constitutes a significant advantage overimmunoassays in which the results are conveyed simply by means ofchanges in color intensity. Another advantage of the subject inventionis the formation of a ring pattern, the combination of ring patternformation and color formation being much easier to recognize than justthe formation of a simple colored spot; this advantage is especiallyimportant when the color is of low intensity. Interpreting changes incolor intensity requires complex optical instruments, or reliance oncrude estimates. Since the subject invention does not require complexequipment, assays may be performed in environments such as a physician'soffice or the field, thus providing for significant savings in both costand time.

The subject invention also provides for a collector-diluter-dispenserapparatus that accurately dilutes a quantity of sample to apredetermined ratio, and permits the diluted sampled to be dispensedinto the assay apparatus. The collector-diluter-dispenser serves tominimize the amount of equipment and steps required to prepare a dilutedsample for testing. Thus the collector-diluter-dispenser apparatusserves to expedite the entire assay procedure and decrease the risk oferror.

The following example is offered by way of illustration and not by wayof limitation

EXAMPLE HTLV-1/2 STAT Test

A diagnostic test employing the subject invention is provided for in acartridge form called the HTLV-1/2 STAT test. The cartridges areprovided in a kit form, including all reagents required for theperformance of an assay, collector-diluter-dispensers, and a tray forholding multiple cartridges.

The HTLV-1/2 STAT test is a rapid qualitative immunoassay for thedetection of IgG antibodies to HTLV-1 and HTLV-2 in human serum, plasma,or whole blood. The test is designed primarily for use with freshsamples.

Principle of the Assay

The HTLV-1/2-STAT test is an easy to use panel test which allows thedetection on a single clinical sample of anti-HTLV-1 and anti-HTLV-2antibodies in comparison with built in positive and negative controls.

Several reactive membrane regions offset by non-porous dividers("miniwells") are divided out on a test cartridge.

These miniwells consist, from top to bottom, of the positive andnegative procedural controls and of an HTLV-1/HTLV-2 viral lysateantigen circle and of an HTLV-1/HTLV-2 synthetic peptide circle. Thefirst miniwell is separated from the second miniwell by a red non-porousdivider. The second and third miniwells are separated by a yellowdivider. Similarly, the third and fourth miniwells are separated by awhite divider.

A single dilution of clinical sample is added to the cartridge. BoundIgG is detected by means of an immunoenzymatic reaction (including anamplification step to provide a high sensitivity) which results in thedevelopment of a stable blue color on the membrane.

A distinctive double ring pattern ensures an easy and sensitive readingof the test results. The positive procedural control verifies that theclinical sample was not omitted, that all reagents were active, and thatthe test was performed correctly.

The negative procedural control allows an easy differentiation of lowpositive specimens versus negative specimens.

No specialized equipment is required to perform or read the test.Results are obtained within a few minutes. The format of the test isadapted to both single testing and batch testing using a standardmultichannel micropipette.

    ______________________________________                                        Kit Components                                                                ______________________________________                                        1.      HTLV-STAT cartridges ready for use.                                   2.      LYOPHILIZED REAGENT (A)                                                       (0.1 M PBS pH 7.4 with 1% casein                                              (w/v), 0.1% Tween 20, v/v)                                            3.      Collector-diluter-dispensers                                          4.      LYOPHILIZED REAGENT (C)                                                       (anti-human IgG Biotin conjugate                                              diluted 1:100 in 0.1 M PBS pH 7.4                                             with 1% casein (w/v)                                                  5.      LYOPHILIZED REAGENT (D)                                                       (alkaline phosphatase streptavidin                                            conjugate diluted 1:100 in 0.1 M                                              borate pH 8 with 1% BSA, w/v).                                        6.      WASH SOLUTION/BUFFER DILUENT (E)                                              (0.05 M borate pH 8 with 0.01%                                                thimerosal w/v).                                                      7.      CHROMOGENIC SUBSTRATE (F)                                                     Bromochloroindoxyl phosphate solution.                                8.      STOPPING SOLUTION (G)                                                         0.2 M phosphate pH 6.                                                 9.      ANTI-HTLV-1 POSITIVE SERUM                                                    (INACTIVATED) (I)                                                     10.     STAT TEST UNIT HOLDING TRAY                                           ______________________________________                                    

(Using a nylon filter and nylon nib (Nib 99356) a solution of HTLV-1peptide at 1 mg/ml DMSO is diluted 1:40 into 0.1M acetate, pH 4. The nibis attached to a reservoir and the nib pressed down onto the nylonfilter until liquid diffuses to cover the miniwell. Two nibs may be usedto form two circles per miniwell.)

Directions for performing the assay are as follows:

Preparation of Reagents

Bring the reagents to room temperature before use. Reconstitute thelyophilized reagents A, C and D with 7 ml of solution E. Gently agitatethe reagents to mix. Allow the reagents to sit at room temperature for afew minutes. Reagent containers are dropper-top bottles. The droppershave measuring lines for adding predetermined amounts of reagent to theassay. If batch testing is to be performed, standard multichannel (8 or12) micropipettes may be used instead of droppers.

Assay Procedure

Label the necessary number of HTLV-STAT cartridges (one for eachspecimen). Place the cartridges (1 to 12) in the device tray.

Add 400 μl of reconstituted reagent A (using the dropper A filled up tothe measuring line) to the HTLV-STAT cartridge. Allow to draincompletely.

Collect and dilute the specimen using a collector-diluter-dispenser(CDD). (The CDD has a nib with a capacity of about 30 μl and a vial with1 ml of 0.1M carbonate/bicarbonate buffer, 1% casein, 0.1% Tween-20,0.01% thimerosal, pH 9.5.)

Using the collector-diluter-dispenser, add 8 drops of diluted specimento the cartridge. Wait 3 to 5 minutes.

Add 200 μl of reconstituted reagent C to the cartridge. Allow to draincompletely.

Add 200 μl of reconstituted reagent D to the cartridge. Allow to draincompletely.

Add 1 ml of wash solution E to the cartridge. Allow to drain completely.

Add 200 ml (8 drops) of chromogenic substrate F to the cartridge.

Wait 3 minutes. Read the results.

Optional: Read the results again 5 to 10 minutes later. Add 200 μl ofstopping solution G to the cartridge.

Interpretation of Results

Validation of the test: blue rings are seen in the first miniwell(positive procedural control) below the red divider. The second miniwell(second miniwell; below the first yellow divider) shows either nothingor light blue rings.

Blue rings in the third and fourth miniwells (between the yellow and thewhite divider) darker than in the second miniwell indicate a positiveresult for anti-HTLV-1 or anti-HTLV-2 antibodies.

Blue color development in all the miniwells indicates a testmalfunction.

Blue rings in the third miniwell with no blue rings in the fourthminiwell or vice versa should be retested and confirmed using othertests.

Used cartridges can be stored desiccated after the reaction is stoppedto serve as a permanent record.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A diagnostic device for measuring an analytewhich is a member of a specific binding pair, where a label is employedfor said measuring which label provides for a visual detectable signal,said device comprising:a housing comprising an upper reservoir portionand a lower casing portion for holding at least one layer; in saidcasing in the direction of flow: a porous reactive filter as the floorof said reservoir component comprising a measurement circle of a memberof a specific binding pair immobilized to said filter and defining aconcentration gradient comprising an inner circle at an elevatedconcentration and an outer contiguous circle at a substantially lowerconcentration; and fluid receiving means for receiving fluid from saidporous reactive filter.
 2. A device according to claim 1, wherein saidfluid receiving means comprises:a porous separation layer; a flowratecontrol layer for reducing the rate of flow; and an absorbent wastefluid receiving layer.
 3. A device according to claim 1, wherein saidporous reactive filter is divided into a plurality of regions separatedby non-porous dividers, wherein at least two of said regions comprise atleast one said measurement circle.
 4. A device according to claim 3,wherein at least one of said regions comprises two said measurementcircles.
 5. A device according to claim 1, wherein said housing is amolded plastic housing.
 6. A device according to claim 1, wherein theaverage concentration of said inner circle is at least two fold greaterthan the highest concentration in the outer circle.
 7. A diagnosticdevice for measuring an analyte comprising a porous reactive filtercomprising at least one region and in at least one of said regions, ameasurement circle of a member of a specific binding pair immobilized tosaid filter and defining a concentration gradient comprising an innercircle at an elevated concentration and an outer contiguous circle at asubstantially lower concentration.
 8. A diagnostic device according toclaim 7, wherein said device comprises at least two regions separated bya non-porous divider.
 9. A diagnostic device according to claim 7,wherein at least one said region comprises at least two measurementcircles having the same specific binding pair member.
 10. A diagnosticdevice according to claim 7, wherein said porous reactive filter isfilter paper.